Biomedical subjects

W Dowling

Publications and source records attributed to W Dowling.

6 recordsLinked to original sources

PubMed2001

VP4 differentially regulates TRAF2 signaling, disengaging JNK activation while directing NF-kappa B to effect rotavirus-specific cellular responses.

Rotaviruses rapidly activate NF-kappaB and induce the secretion of selected chemokines after infection. The ability of rotavirus particles lacking genomic RNA to activate NF-kappaB suggested that rotavirus proteins direct cell signaling responses. We identified conserved TNFR-associated factor (TRAF) binding motifs within the rotavirus capsid protein VP4 and its N-terminal VP8* cleavage product. TRAFs (-1, -2, and -3) are bound by the rhesus rotavirus VP8* protein through three discrete TRAF binding domains. Expression of VP4 or VP8* from rhesus or human rotaviruses induced a 5-7-fold increase in NF-kappaB activity and synergistically enhanced TRAF2-mediated NF-kappaB activation. Mutagenesis of VP8* TRAF binding motifs abolished VP8* binding to TRAFs and the ability of the protein to activate NF-kappaB. Expression of pathway-specific dominant negative (DN) inhibitors DN-TRAF2 or DN-NF-kappaB-inducing kinase also abolished VP8*-, VP4-, or rotavirus-mediated NF-kappaB activation. These findings demonstrate that rotavirus primarily activates NF-kappaB through a TRAF2-NF-kappaB-inducing kinase signaling pathway and that VP4 and VP8* proteins direct pathway activation through interactions with cellular TRAFs. In contrast, transcriptional responses from AP-1 reporters were inhibited 5-fold by VP8* and were not activated by rotavirus infection, suggesting the differential regulation of TRAF2 signaling responses by VP8*. VP8* blocked JNK activation directed by TRAF2 or TRAF5 but had no effect on JNK activation directed by TRAF6 or MEKK1. This establishes that fully cytoplasmic rotaviruses selectively engage signaling pathways, which regulate cellular transcriptional responses. These findings also demonstrate that TRAF2 interactions can disengage JNK signaling from NF-kappaB activation and thereby provide a new means for TRAF2 interactions to determine pathway-specific responses.

Animals↗

PubMed2000

Selective membrane permeabilization by the rotavirus VP5* protein is abrogated by mutations in an internal hydrophobic domain.

Rotavirus infectivity is dependent on the proteolytic cleavage of the VP4 spike protein into VP8* and VP5* proteins. Proteolytically activated virus, as well as expressed VP5*, permeabilizes membranes, suggesting that cleavage exposes a membrane-interactive domain of VP5* which effects rapid viral entry. The VP5* protein contains a single long hydrophobic domain (VP5*-HD, residues 385 to 404) at an internal site. In order to address the role of the VP5*-HD in permeabilizing cellular membranes, we analyzed the entry of o-nitrophenyl-beta-D-galactopyranoside (ONPG) into cells induced to express VP5* or mutated VP5* polypeptides. Following IPTG (isopropyl-beta-D-thiogalactopyranoside) induction, VP5* and VP5* truncations containing the VP5*-HD permeabilized cells to the entry and cleavage of ONPG, while VP8* and control proteins had no effect on cellular permeability. Expression of VP5* deletions containing residues 265 to 474 or 265 to 404 permeabilized cells; however, C-terminal truncations which remove the conserved GGA (residues 399 to 401) within the HD abolished membrane permeability. Site-directed mutagenesis of the VP5-HD further demonstrated a requirement for residues within the HD for VP5*-induced membrane permeability. Functional analysis of mutant VP5*s indicate that conserved glycines within the HD are required and suggest that a random coiled structure rather than the strictly hydrophobic character of the domain is required for permeability. Expressed VP5* did not alter bacterial growth kinetics or lyse bacteria following induction. Instead, VP5*-mediated size-selective membrane permeability, releasing 376-Da carboxyfluorescein but not 4-kDa fluorescein isothiocyanate-dextran from preloaded liposomes. These findings suggest that the fundamental role for VP5* in the rotavirus entry process may be to expose triple-layered particles to low [Ca](i), which uncoats the virus, rather than to effect the detergent-like lysis of early endosomal membranes.

Amino Acid Motifs↗

PubMed1999

Rotavirus capsid protein VP5* permeabilizes membranes.

Proteolytic cleavage of the VP4 outer capsid spike protein into VP8* and VP5* proteins is required for rotavirus infectivity and for rotavirus-induced membrane permeability. In this study we addressed the function of the VP5* cleavage fragment in permeabilizing membranes. Expressed VP5* and truncated VP5* proteins were purified by nickel affinity chromatography and assayed for their ability to permeabilize large unilamellar vesicles (LUVs) preloaded with carboxyfluorescein (CF). VP5* and VP5* truncations, but not VP4 or VP8*, permeabilized LUVs as measured by fluorescence dequenching of released CF. Similar to virus-induced CF release, VP5*-induced CF release was concentration and temperature dependent, with a pH optimum of 7.35 at 37 degrees C, but independent of the presence of divalent cations or cholesterol. VP5*-induced permeability was completely inhibited by VP5*-specific neutralizing monoclonal antibodies (2G4, M2, or M7) which recognize conformational epitopes on VP5* but was not inhibited by VP8*-specific neutralizing antibodies. In addition, N-terminal and C-terminal VP5* truncations including residues 265 to 474 are capable of permeabilizing LUVs. These findings demonstrate that VP5* permeabilizes membranes in the absence of other rotavirus proteins and that membrane-permeabilizing VP5* truncations contain the putative fusion region within predicted virion surface domains. The ability of recombinant expressed VP5* to permeabilize membranes should permit us to functionally define requirements for VP5*-membrane interactions. These findings indicate that VP5* is a specific membrane-permeabilizing capsid protein which is likely to play a role in the cellular entry of rotaviruses.

Animals↗

PubMed1993

A community strategic planning process for elder services.

Dynamic changes in population, patient demographics, and healthcare financing mechanisms create shifting service priorities. Strategic planning has become a vitally important skill for nurse executives, who must extend their efforts and influence beyond traditional agency boundaries. The authors present an application of the strategic planning process as it was used to develop recommendations for eldercare services in one southwestern, urban community.

Adult↗

PubMed1993

Integrating strategic planning and quality management in a multi-institutional system.

Integrating total quality management (TQM) into the strategic planning process of a multi-institutional health system is a high priority. Through the strategic plan, system leadership gives TQM concepts prominence and visibility and provides strategic directions for their implementation. The Sisters of Providence Health System recently developed an approach to integrating TQM into its System Strategic Plan. Implementation priorities include more formalized direction for TQM by the system board, intensive TQM educational programs, greater consistency of quality information and outcome measures, and more focused attention to patient needs.

Catholicism↗

PubMed1987

Dimensions of religiosity in old age: accounting for variation in types of participation.

Research on religiosity and aging has focused more on church attendance than on the religious role and the strength of beliefs and the extent of private devotions. This study of 123 older West Texans examines the degree to which nine variables account for variation in both organized religious activities and private religious behavior. These include three social activity and interaction variables, three religion variables, and three personal variables (health, age, and income). Strength of religious conviction proved to be the strongest predictor of both types of religious participation. Multiple-regression analysis did not support the hypothesis that private religious activity is compensatory; that is, it does not make up for increased social or personal deprivation. Poor health, low income, reduced activity, and living alone did not predict higher levels of nonorganized religious behavior. However, strong kin/friend networks did predict high levels of private devotion. This research suggests that frequent interaction in a social network contributes to the spiritual well-being of elderly persons; that is, it affirms the wholeness of their lives.

Age Factors↗